Voltage-activated Calcium Currents in Octopus Cells of the Mouse Cochlear Nucleus
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Octopus cells, neurons in the most posterior and dorsal part of the mammalian ventral cochlear nucleus, convey the timing of synchronous firing of auditory nerve fibers to targets in the contralateral superior paraolivary nucleus and ventral nucleus of the lateral lemniscus. The low input resistances and short time constants at rest that arise from the partial activation of a large, low-voltage-activated K+ conductance (gKL) and a large mixed-cation, hyperpolarization-activated conductance (gh) enable octopus cells to detect coincident firing of auditory nerve fibers with exceptional temporal precision. Octopus cells fire conventional, Na+ action potentials but a voltage-sensitive Ca2+ conductance was also detected. In this study, we explore the nature of that calcium conductance under voltage-clamp. Currents, carried by Ca2+ or Ba2+ and blocked by 0.4 mM Cd2+, were activated by depolarizations positive to −50 mV and peaked at −23 mV. At −23 mV they reached 1.1 ± 0.1 nA in the presence of 5 mM Ca2+ and 1.6 ± 0.1 nA in 5 mM Ba2+. Ten micromolar BAY K 8644, an agonist of high-voltage-activated L-type channels, enhanced IBa by 63 ± 11% (n = 8) and 150 μM nifedipine, an antagonist of L-type channels, reduced the IBa by 65 ± 5% (n = 5). Meanwhile, 0.5 μM ω-Agatoxin IVA, an antagonist of P/Q-type channels, or 1 μM ω-conotoxin GVIA, an antagonist of N-type channels, suppressed IBa by 15 ± 4% (n = 5) and 9 ± 4% (n = 5), respectively. On average 16% of the current remained in the presence of the cocktail of blockers, indicative of the presence of R-type channels. Together these experiments show that octopus cells have a depolarization-sensitive gCa that is largely formed from L-type Ca2+ channels and that P/Q-, N-, and R-type channels are expressed at lower levels in octopus cells.
Keywordsvoltage-sensitive calcium channels cochlear nucleus hearing patch clamp brain slices
Octopus cells are remarkable neurons. Their time constants are among the fastest that have been recorded in the mammalian nervous system. Low input resistances allow octopus cells to generate exceptionally small, brief, and sharply timed excitatory postsynaptic potentials (EPSPs). Octopus cells detect the coincidence of many small subthreshold synaptic inputs from individual auditory nerve fibers (Golding et al. 1995; Ferragamo and Oertel 2002; McGinley and Oertel 2006). The jitter in the timing of firing in response to sounds is less than 200 μs (Oertel et al. 2000). Octopus cells are exceptional not only in the temporal precision with which they encode acoustic information but also in the firing rates they can sustain. For example, in responses to periodic stimuli octopus cells can fire at every cycle to about 800 Hz (Rhode and Smith 1986).
To understand how octopus cells can signal with such temporal precision, we have undertaken a study of their biophysical characteristics. Whole-cell patch recordings in the current-clamp mode from cell bodies revealed that octopus cells have exceptionally strong hyperpolarization-activated inward and repolarizing outward currents as well as Ca2+-dependent and Na+-dependent, tetrodotoxin (TTX)-sensitive action potentials (Golding et al. 1999). The hyperpolarization-activated mixed cation conductance, gh, and the low-voltage- and high-voltage-activated K+ conductances, gKL and gKH, were studied under voltage-clamp (Bal and Oertel 2000; Bal and Oertel 2001). The TTX-sensitive inward current, however, was generally not possible to clamp; in the absence of TTX, depolarizing voltage pulses produce escaped action potentials. The low-voltage-activated, repolarizing K+ conductance that is partially activated at rest makes octopus cells sensitive to the rate at which they are depolarized (Ferragamo and Oertel 2002). The present study is of the depolarization-activated Ca2+ inward current under voltage-clamp.
Voltage-dependent Ca2+ channels fall into three families that differ in the voltage range of activation, the rate at which they deactivate; in the voltage range, rate, and extent of inactivation; and in their sensitivity to pharmacological agents (Tsien et al. 1988; Bean 1989; Hille 2001). Most L-type calcium channels that are encoded by genes in the CaV1 family are activated by large depolarizations, more positive than −30 mV; CaV1.3α channels are exceptional in that they are activated by lower voltages, positive to −65 mV (Xu and Lipscombe 2001; Lipscombe et al. 2004). L-type channels are sensitive to dihydropyridines. They are enhanced by the dihydropyridine agonist, BAY K 8644, and blocked by the antagonist, nifedipine (Fox et al. 1987b; Carbone and Swandulla 1989). The sensitivity to dihydropyridine antagonists varies over an order of magnitude, however (Kanngiesser et al. 1988; Xu and Lipscombe 2001). P/Q-, N-, R-type channels are encoded by CaV2.1, 2.2, and 2.3, respectively. P/Q- and N-type calcium channels are sensitive to ω-agatoxin IVA and ω-conotoxin GVIA, respectively (McCleskey et al. 1987; Mintz et al. 1992; Boland et al. 1994; Overholt and Prabhakar 1997; Martinez-Pinna et al. 2002). P/Q- and N-type channels are activated by large depolarizations and inactivate rapidly but only partially. The R-type current requires large depolarizations, is insensitive to dihydropyridines, ω-agatoxin, and ω-conotoxin, and is rapidly inactivating (Randall and Tsien 1995). The low-voltage-activated T-type channels are encoded by genes in the CaV3 family and inactivate rapidly and completely. Diversity in voltage-gated Ca2+ channels arises not only from their being encoded by various families of genes but also because the transcripts from those genes have numerous alternatively spliced forms (Lipscombe et al. 2002).
The results of this study demonstrate that L-type Ca2+ channels predominate in octopus cells. More than 60% of the current carried by Ba2+ is sensitive to blockers of L-type channels but N-, P/Q-, and R-type channels were also detected. What the role of the voltage-sensitive Ca2+ current is in octopus cells is not entirely clear; it might serve to monitor electrical activity in octopus cells and to regulate homeostatic mechanisms.
Brain slice preparation
Coronal slices of the ventral cochlear nucleus (VCN) were prepared from mice (ICR strain) of between 17 and 19 postnatal days. The animals were decapitated and the head was immersed in normal physiological saline. After the removal of the brain from the skull, it was cut coronally at the midcollicular level. The rostral surface of the caudal part of the brain was mounted on a Teflon block with a cyanoacrylate glue (Superglue). Slices of 180 μm in thickness were cut using an oscillating tissue slicer (Frederick Haer, Newbrunswick, ME, USA). Slices were transferred to a storage chamber containing fresh, oxygenated, physiological saline at 33°C to allow slices to recover for up to 2 h. For recording, the slices were transferred to a chamber of about 0.3 ml through which oxygenated saline was continuously perfused at about 5 ml/min. The temperature was kept at 33°C with a custom-made feedback-controlled heater.
Identification of octopus cells
Cells in slices were visualized with differential interference contrast optics (Axioscope FS, Zeiss) through a ×63 water immersion objective. Octopus cells were initially identified by their characteristic location in a triangular region of heavily myelinated fibers just ventral and caudal to the translucent granule cell layer that separates the ventral from the dorsal cochlear nucleus. Illumination under bright-field conditions with a field diaphragm that was minimally open made octopus cells appear bright among dark bundles of myelinated fibers. Their characteristic response pattern to injected hyperpolarizing and depolarizing current pulses was used to confirm the visual identification after breaking into octopus cells with patch electrodes (Golding et al. 1995; Golding et al. 1999; Bal and Oertel 2000; Bal and Oertel 2001).
Solutions and drugs
The normal physiological saline contained (in mM): 130 NaCl, 3 KCl, 1.2 KH2PO4, 2.4 CaCl2, 1.3 MgSO4, 20 NaHCO3, 3 HEPES, 10 glucose, saturated with 95% O2/5% CO2, pH 7.4, between 25 and 31°C (Bal and Oertel 2000; Bal and Oertel 2001). Normal physiological saline was used for preparation and incubation of slices. In current-clamp experiments involving the use of Cd2+, the external solution contained (in mM): 138 NaCl, 3 KCl, 2.4 CaCl2, 1.3 MgCl2, 10 HEPES, 10 glucose, pH 7.4. To study Ca2+ conductances under voltage clamp, they were separated from other conductances pharmacologically. The extracellular solution under “control” conditions contained (in mM): 135 tetraethylammonium (TEA) chloride; 10 CsCl, 10 4-aminopyridine (4-AP), 5 CaCl2 or 5 BaCl2, 10 HEPES, 10 glucose, 1 μM TTX, 40 μM 6,7-dinitroquinoxaline-2,3-dione (DNQX), pH 7.4. For measurements of barium currents through calcium channels, CaCl2 was replaced by BaCl2 on an equimolar basis. Cesium was used to suppress the hyperpolarization-activated current, Ih (Bal and Oertel 2000), TEA and 4-AP to block K+ currents (Bal and Oertel 2001), 1 μM TTX to block voltage-sensitive Na+ current (Golding et al. 1999), and 40 μM DNQX to block synaptic currents (Tocris Cookson, UK). Test solutions were applied to the chamber by redirecting the flow of liquid through a system of tubing and valves.
To minimize the errors introduced by series resistance, only pipettes of low resistance (3–4 MΩ) were used. They were pulled from borosilicate glass (1.2 mm OD) and were filled with a solution that contained (in mM): 110 potassium gluconate, 9 HEPES, 9 EGTA, 4.5 MgCl2, 14 phosphocreatinine (Tris salt), 4 ATP (Na-salt), and 0.3 GTP (Tris salt); pH was adjusted to 7.4 with KOH (Forscher and Oxford 1985). TTX, α-dentrotoxin (α-DTX), Bay K 8644, ω-agatoxin IVA, and ω-conotoxin GVIA were purchased from Alomone labs (Israel) and other chemicals were obtained from Sigma. Bay K 8644 and nifedipine were dissolved in dimethylsulfoxide (DMSO) to make stock solutions and stored in single-use-aliquots in the dark at −20°C. The final DMSO concentration was 0.01% and in separate control experiments was found to have no effect on the cells’ calcium currents. TTX, α-DTX, ω-agatoxin IVA, and ω-conotoxin GVIA were dissolved in distilled water to make stock solutions and stored in single-use-aliquots at −20°C. The doses were close to saturating for each of the calcium channel blockers.
Whole-cell recordings and data acquisition
Current- and voltage-clamp recordings were performed with standard whole-cell patch-clamp techniques using an Axopatch-200A amplifier. Data were low-pass-filtered at 5–10 kHz. Current and voltage records were sampled at 10–40 kHz and were digitized online using a Digidata 1320 interface (Axon Instruments, Foster City, CA, USA) and fed both to a chart recorder and to an IBM-compatible personal computer for storage and further analyses. Stimulus generation, data acquisition, and offline analysis of digitized data were done using pClamp software (version 8.03; Axon Instruments). After the formation of high-resistance seals (>1 GΩ) negative pressure was applied to obtain the whole-cell configuration. Series resistance varied from 6 to15 MΩ. All reported results were from recordings in which approximately 85–90% of the series resistance could be compensated on line. All recordings have been compensated for a junction potential of −12 mV. Data are expressed as mean ± SE, with n being the number of cells in which the measurement was made.
The present study is based on whole-cell recordings from 78 octopus cells that lasted between 20 min and 2 h. The characteristics of octopus cells have been described in previous reports (Golding et al. 1999; Bal and Oertel 2000; Bal and Oertel 2001; Cao and Oertel 2005).
Voltage dependence of activation
Also in contrast with Ih, IKL, and IKH, ICa was difficult to clamp even when other conductances were largely blocked. Good voltage control of calcium currents was obtained from only about one in ten recorded octopus cells. Figure 2A, B shows an example of an octopus cell in which ICa could be clamped; in this cell the peak inward current rose gradually over a range of depolarizing voltage steps. Figure 2C, D shows two recordings from octopus cells in which calcium currents could not be clamped. In these cells the all-or-none current peaks were indicative of escaped action potentials. Figure 2C illustrates how step depolarization to levels more positive than −45 mV evoked obviously escaped action potentials. The I/V plot in Figure 2D shows that the peak inward current rose in an all-or-none step, also at −45 mV. Escape from voltage control could have arisen from imperfect space-clamp or from a voltage-clamp that was slow relative to the kinetics of the current or probably from a combination of the two factors. Octopus cells have dendrites that are about one length constant long (McGinley et al. 2005). They also have large axons with local collaterals and terminals whose position and length is variable (Golding et al. 1995). Anatomical labeling has shown that in many cases the axons of octopus cells are cut close to the cell body and lack axon terminals (Oertel et al. 1990; Golding et al. 1995). If all gCa were in terminals, we would have expected some octopus cells to have little or no gCa but that was not the case; every cell we tested had gCa. We conclude, therefore, that at least some gCa is associated with the cell body and dendrites.
Voltage dependence of inactivation
Octopus cells have little or no low-voltage-activated Ca2+ current. T-type currents activate at relatively hyperpolarized potentials, near −70 mV, and are strongly inactivated; some L-type currents also activate at relatively hyperpolarized potentials (Xu and Lipscombe 2001). IBa failed to activate between −70 and −50 mV even when the holding potential was −90 mV, indicating that little, if any, of IBa was mediated through low-voltage-activated L-type or T-type Ca2+ channels.
To identify what type(s) of calcium channels underlies the macroscopic current, its sensitivity was tested to a variety of Ca2+ channel agonists and antagonists. As noted above, ICa and IBa in octopus cells were completely blocked by 0.4 mM Cd2+, a nonspecific calcium channel blocker (Figs. 2 and 3).
P/Q- and N-type calcium channels are sensitive to ω-agatoxin IVA and ω-conotoxin GVIA, respectively (McCleskey et al. 1987; Mintz et al. 1992; Boland et al. 1994). The peak IBa was reduced by 15 ± 4% (n = 5) by 0.5 μM ω-agatoxin IVA (Fig. 6C). Application of 1 μM ω-conotoxin GVIA reduced IBa by 9 ± 4% (n = 5) (Fig. 6D). These results indicate that P/Q- and N-type channels each contribute a relatively small fraction of gCa. The currents that are sensitive to dihydropyridines, ω-agatoxin IVA, and ω-conotoxin GVIA begin to be activated at about −50 mV and peak near −25 mV, as expected for high-voltage-activated ICa (Fig. 6B–D).
Octopus cells possess voltage-sensitive Ca2+ currents. In current-clamp, Golding et al. (1999) showed that regenerative, Ca2+-mediated action potentials could be evoked in octopus cells when Na+ and K+ currents were blocked. Now under voltage-clamp we have shown that Ca2+ currents are mediated through several types of Ca2+ channels. The Ca2+ currents were activated by depolarizations to membrane potentials more positive than −50 mV, indicating that they are high-voltage-activated. When measured in the presence of 5 mM extracellular Ca2+, the peak current was about 1.1 nA at −23 mV; in the presence of 5 mM extracellular Ba2+ the peak current was about 1.6 nA. The sensitivity of these currents to agonists and antagonists provided clues about the types of ion channels that mediate them. The sensitivity to BAY K 8644 and nifedipine indicates that about 65% of the conductance is through L-type Ca2+ channels. The sensitivities of 15% of IBa to 0.5 μM ω-agatoxin IVA and 9% to 1 μM ω-conotoxin GVIA indicate that a smaller proportion of the current was mediated through P/Q- and N-type channels, respectively. The insensitivity of about 16% of the current to any of these blockers suggests that octopus cells also have R-type channels. In contrast to recordings of IKL, IKH, and Ih, which could be well clamped in most octopus cells, ICa could be clamped in only about 10% of octopus cells. Either the Ca2+ conductances reside in regions of the cell that are electrically distant and/or that the voltage clamp was not fast enough to clamp the rapid regenerative currents. Some of the current must have entered through the soma and/or dendrites, however, because Ca2+ currents were observed in every octopus cell and we know that axons of octopus cells in coronal slices are often cut near the soma (McGinley et al. 2005).
Functional role of voltage-sensitive calcium currents in signaling by octopus cells
There are about 200 octopus cells in each posterior VCN of a mouse (Osen 1969; Willott and Bross 1990) that project contralaterally through the intermediate acoustic stria to the superior paraolivary nucleus and to the ventral nucleus of the lateral lemniscus (Warr 1969; Smith et al. 1993; Schofield 1995; Adams 1997; Vater et al. 1997). The pathway through octopus cells may be involved in the analysis of temporal features of signals for the recognition of natural sounds (Covey and Casseday 1991; Oertel and Wickesberg 2002). Octopus cells are broadly tuned and are driven at the onset of tones and noise and by click trains (Godfrey et al. 1975; Rhode et al. 1983; Rhode and Smith 1986; Oertel et al. 2000; Smith et al. 2005). Of all cell types in the VCN, they respond with the strongest synchronization to amplitude-modulated sounds and to simple speech sounds (Rhode 1994; Rhode 1998). The jitter in the timing of firing in response to trains of clicks is less than 200 μs (Oertel et al. 2000). In responses to periodic stimuli octopus cells can fire at every cycle to about 800 Hz (Rhode and Smith 1986).
Octopus cells detect synchronous firing among auditory nerve fibers and convey its occurrence with action potentials that are generated with temporal precision (Golding et al. 1995; Oertel et al. 2000; Ferragamo and Oertel 2002; McGinley and Oertel 2006). Two opposing voltage-sensitive conductances, gKL and gh, are activated at rest and endow octopus cells with low input resistances (6 MΩ), short time constants (∼200 μs), and consequently brief EPSPs (∼1 ms) (Golding et al. 1995; Gardner et al. 1999; Bal and Oertel 2000; Bal and Oertel 2001; Gardner et al. 2001; Cao and Oertel 2005). Sensitivity to the rate of depolarization requires that EPSPs sum on their rising phases and make coincidence detection yet sharper (Ferragamo and Oertel 2002; McGinley and Oertel 2006). The high-voltage-activated K+ conductance, gKH, presumably contributes to rapid repolarization that allows octopus cells to fire at high frequencies (Bal and Oertel 2001; Macica et al. 2003). Coincident activation of small synaptic currents from large numbers of auditory nerve fibers depolarize the dendrites and cell bodies of octopus cells and can bring the axon to threshold (Golding et al. 1995; Golding et al. 1999).
Action potentials are conventional in that they are all-or-none and sensitive to TTX (Golding et al. 1995; Golding et al. 1999; Ferragamo and Oertel 2002). Voltage-gated Ca2+ currents will be activated by excitatory synaptic potentials and by regenerative Na+ action potentials that depolarize octopus cells but those currents are small at the cell body. ICa reaches maximally 1.1 nA at −23 mV; at this same voltage IKL is estimated to be about 30 nA (Bal and Oertel 2001). Furthermore, ICa is slow so that less than the maximal current would be expected to be activated by EPSPs or action potentials: at −23 mV, ICa reaches its peak about 5 ms after the onset of a square pulse of voltage whereas depolarizing responses to activation by the auditory nerve reach a peak within about 0.5 ms. Being small relative to other voltage-sensitive currents and being slow relative to the duration of voltage changes in octopus cells, the contribution of voltage-gated Ca2+ currents to electrical signaling at the cell body is probably minor.
It is possible that the entry of calcium serves as a measure of activity and drives homeostatic mechanisms either at the cell body or elsewhere in octopus cells. L-type calcium channels are widespread, perhaps even ubiquitous, in the central nervous system and regulate protein phosphorylation, neuronal plasticity, and gene expression in neuronal cells (Murphy et al. 1991; Hell et al. 1993; Misra et al. 1994; Finkbeiner and Greenberg 1998). Calcium enters octopus cells not only through voltage-gated but also through ligand-gated receptors. Glutamate receptors in octopus cells do not contain GluR2 subunits and are calcium-permeable (Gardner et al. 1999; Gardner et al. 2001). The calcium from voltage- and ligand-gated ion channels is likely to affect different locations, however. Octopus cells contain relatively high levels of the calcium-buffering protein, calretinin, which presumably localizes calcium transients to the vicinity of their sources (Adams 1997). L-type calcium channels have the most rapid activation kinetics, the least voltage-dependent inactivation, and the most calcium-dependent inactivation of the high-voltage-activated calcium channels. Large EPSPs that signal the coincident firing of many auditory nerve fibers activate those L-type calcium channels and promote the entry of calcium. Whether they arise through activation of calcium-permeable glutamate receptors or L-type channels, high levels of calcium should inactivate the L-type channels and reduce further calcium entry.
We are grateful for the help and support of our colleagues Xiao-Jie Cao, Aldo Rodrigues, Shalini Shatadal, and Ravi Kochhar. An anonymous reviewer also made significant contributions. This work was supported by a grant from NIH DC00176.
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